Summary: Information regarding the minimal requirements for activation of primed T cells is readily available. However, the immune system is most amenable to modulation before completion of antigen priming. This project is to define the requirements for activation of unprimed naive T cells and to evaluate the effects of additional stimuli on the magnitude and type of T cell activation elicited. With the use of a potent antigenic peptide-MHC complex, we have been successful in the activation of phenotypically naive CD8+ transgenic T-cells. These T-cells have been stimulated to proliferate, secrete IL-2 and mature to cytolytic effectors in the absence of additional signals from antigen presenting cells. Through the use of this experimental system and purified recombinant soluble ICAM-1, we have evaluated the effects on T-cell activation of adding an isolated costimulatory signal. ICAM-1 can increase proliferation and cytokine production of CD8+ T-cells with under varying levels TCR signal intensity. We have also demonstrated that ICAM-1 has a differential effect on CD4+ and CD8+ T-cells and that the ICAM-1 enhancement of CD8+ T-cell activation can occur in the absence of an organized immunological synapse. Evaluation of other purified adhesion/costimulatory molecules may reveal different effects. To expand our readout of T-cell activation, we have added methodologies that have greater relevance to the in vivo outcome of T-cell activation. The use of transgenic T-cells in an adoptive transfer model has demonstrated the criticality of T-cell localization in the type of T-cell activation observed. This suggests the tissue distribution of immunomodulatory agents may have unexpected consequences. We have also evaluated microscopic changes in T-cells by studying the movement of the cell-surface mucin molecule CD43 after T-cell activation. These experiments have allowed us to assess subtle changes in T-cell activation. Using this system, we have been able to demonstrate immunologic effects of a statin HMG CoA reductase inhibitor. Although immunologic mechanisms have been suggested as a reason for the unexplained benefits of statins, our assay system demonstrates a specific microscopic readout for a statin effect on T-cells. This type of assay may facilitate comparisons of different statins for immunomodulatory effects. This system will allow us to evaluate specific changes in naive T cell responses caused by the deletion or addition of lymphokines, adhesion molecules or costimulatory molecules. This can be done in the absence of unknown contributions from antigen presenting cells and correlated with cellular morphology and in vivo data using the same transgenic T-cells used in vitro. These results can be correlated with biochemical data on the involved molecules such as binding constants and thermodynamics by using SPR technology. We have performed this type of biochemical analysis on domains of the antigen presenting MHC1 molecule. The molecular requirements for T-cell activation and the varying phenotypes of this activation are critical for the generation of successful immunity and the modulation of aberrant immune responses. Understanding T-cell activation and how it can be modulated is important in assessing the mechanism of action of therapeutic antibodies directed against T-cells.